PROJECT 4: Genetic and Structural Basis for Human Antibody Inhibition of Dengue Viruses (Vanderbilt University) SUMMARY One of the primary goals of this P01 program project proposal is to define the functional and antigenic specificity of the memory humoral response before and after dengue virus (DENV) infection. Here, we will determine the molecular and structural basis for type-specific and cross-reactive human B cell responses to DENV infection. Aim 1 of Project 4 will study the epitopes and structures recognized by DENV3 type-specific potently neutralizing antibodies (Abs). The goal is to define a comprehensive antigenic map of neutralizing determinants on the DENV E protein, which will inform DENV vaccine design and testing efforts. In past work with investigators in this consortium, we have mapped many of the major antigenic determinants on DENV1 and DENV2. The protective determinants on DENV3 are less well studied. Fortunately, in preliminary experiments, in collaboration with Project 1 and Core C, we have generated a significant panel of potent DENV3-specific neutralizing monoclonal Abs (mAbs) from the Nicaraguan Pediatric Dengue Cohort Study (PDCS) samples. It is clear that many of these antibodies recognize novel epitopes that are not previously known. We will use these reagents to identify the molecular and structural basis for recognition using alanine scanning mutagenesis, hydrogen deuterium exchange mass spectrometry, cryo-EM and crystallography studies. If the antigenic maps appear incomplete, as determined by Project 1 in dengue-endemic populations, we will generate additional mAbs from the peripheral blood mononuclear cells (PBMCs) collected from children with documented repeat DENV3 infections, as well as DENV1 and DENV2 infections, in the PDCS to define the complete antigenic landscape. We hypothesize that most DENV3-specific Abs are directed to quaternary epitopes, including those near the DENV envelope domain I/II (EDI/II) hinge region, but that there is a diversity of binding poses and angles for recognition of this complex region. In Aim 2, we will define comprehensive antigenic maps for cross-reactive Abs that recognize and neutralize viruses of all 4 DENV serotypes. In preliminary studies, we have isolated broad and potent mAbs that have distinct profiles from those of the limited number of E dimer epitope (EDE) mAbs reported to date. The new mAbs suggest there are additional sites of vulnerability for broad and potent neutralizing responses that are not yet understood. Studies in this aim will define with biochemical, genetic and structural approaches the novel epitopes associated with highly neutralizing cross-reactive mAbs that differ from EDE epitopes after 2 DENV infection. Finally, in Aim 3, we will use emerging techniques in adaptive immune receptor next generation sequencing to interrogate the Ab variable gene repertoires in PDCS subjects. We will use deep sequencing of peripheral blood Ab gene repertoires in prior (pre-2 infection, pre-vaccination) PBMC samples from the same individuals. These studies will provide a global view of repertoire responses that will complement the molecular ?snapshots? afforded by mAb studies in Aims 1 and 2. The repertoire studies will elucidate the complexity and specificity of the human B cell response to DENV infection on a more systematic level. The P01 integrates the work of several laboratories with expertise in different aspects of DENV biology and adaptive immunity. The human mAbs isolated in Project 4 will be used to study the molecular, structural and genetic basis for neutralizing responses but also will generate and distribute key mAb reagents that facilitate the studies in Projects 1 and 2 in this P01. Chimeric antigens produced on Project 2 will be used to identify subjects with novel B cell responses, and also to map new mAb specificities. The tasks proposed here could not be performed by any single laboratory in the P01 because of the expertise needed in Ab engineering and deep sequence analysis, the scale of production required, and the need for unique dengue reagents, cohort participants, and molecular biology approaches. Understanding human B cell responses to DENV at both the clonal and overall genetic repertoire level promises to give significant new insights into the molecular and genetic basis for type-specific and cross- reactive human B cell responses.